The overall goal of this proposal is to apply several novel techniques to comprehensive quantitative comparisons of human breast cancer protein profiles focusing on changes associated with invasive/ metastatic potential. Our global proteome strategy should result in reliable quantitative comparisons of up to 10,000 proteins instead of the 1,500 to 2,500 protein spots typically detected on high resolution 2D gels. The most novel and key part of this strategy is a new method that we recently developed for prefractionating whole cell extracts prior to 2D PAGE. This method, termed microscale solution IEF (mu sol-IEF), uses small chambers separated by large pore acrylamide discs with immobilines at specific pH's to separate cell extracts into well resolved pools based upon pI's. A second novel feature of our global proteome strategy is use of slightly overlapping (about +/-0.1 pH) custom-made narrow pH range IPG gels tailored to match mu sol-IEF pools, rather than existing commercial 1 pH unit IPG gels with either 0 or 0.5 pH unit overlaps. A third novel feature is use of high-resolution 1D gels coupled with LC- MS-MS to analyze the insoluble proteins and large soluble proteins (100-500+ kDa). These two protein groups represent >25% of total cell protein mass, but are usually ignored in 2D protein profile comparisons. Finally, we recently optimized femtomole in-gel trypsin digestion to improve sensitivity and reliability of MALDI MS and LC-MS/MS identifications of targeted proteins. Our preliminary results show each of these techniques is very promising, but the feasibility of reliably quantitating 7,500 to 10,000+ proteins in human tumor cells remains to be demonstrated. The one year R21 phase will further optimize and integrate these methods to demonstrate proof-of-principle of our integrated global proteome analysis strategy using human breast cancer cells. The three year R33 phase will then apply the optimized global strategy to comparisons of multiple human breast cancer cell lines and tumors to identify protein changes associated with increased invasion and metastatic potential.